PROJECT SUMMARY Methicillin-resistant S. aureus (MRSA) infection complicated by influenza emerges as a leading cause of death during recent influenza pandemics and epidemics. Defective bacterial control and exaggerated lung inflammation are believed to be responsible for high mortality in patients and animals. However, a hitherto incomplete understanding of coinfection pathophysiology has hampered the development of effective treatments. NADPH oxidase 2 (NOX2) is an enzyme complex predominantly expressed by phagocytes. The generation of reactive oxygen species (ROS) through NOX2, a process called oxidative burst, is required for lung S. aureus clearance. During the initial funding period, the work from PI's laboratory has established that intracellular ROS in phagocytes decrease at the recovery stage of influenza infection, thereby leading to defective killing of S. aureus. However, although intracellular ROS are reduced per cell, there are excessive inflammatory cells that release oxidants during coinfection. This event eventually culminates in lethal oxidative lung damage. Furthermore, preliminary studies in PI's laboratory reveal that type I IFN (IFN-I) signaling facilitates monocyte recruitment as it inhibits lung bacterial clearance, whereas IFN-? signaling promotes lung inflammation and lethal lung damage after influenza and S. aureus coinfection. Therefore, it is hypothesized that influenza- induced IFN responses disrupt the balance between oxidative burst-associated antibacterial immunity and inflammation, and results in not only susceptibility to secondary S. aureus infection but also lethal lung injury thereafter. The approaches to test the hypothesis include: 1) to determine whether influenza-induced IFN-I impairs antibacterial immunity by promoting monocyte recruitment. Specifically, the cellular and signaling mechanisms for IFN-I-suppressed bacterial killing will be examined in mouse coinfection models; 2) to elucidate how IFN-? promotes an inflammatory cytokine storm and lethal lung injury during influenza and S. aureus coinfection. Specifically, the contribution of the IFN-?-driven destructive inflammation cascade to lethal lung damage will be determined in a clinical-relevant coinfection mouse model; and 3) refine combination treatment strategies that target both intracellular bacteria and lung inflammation. It has been reported by PI's laboratory that combination treatment with antibiotic and NOX2 inhibitor significantly improves animal survival from influenza and MRSA coinfection. Specific aim 3 is to optimize this combination therapeutic approach based on the findings from studies proposed in aim 1 &2. The ultimate goal of this project is to establish the treatment strategy to restore antimicrobial defense while limiting inflammatory lung damage during influenza and MRSA coinfection.